The spleen has been considered an enigmatic and mysterious organ since its original discovery in the ancient era. The spleen specifically controls phagocytic removal of abnormal red blood cells (RBCs) and particulate matter, iron storage from RBCs, initial immune response to circulating antigens, and fetal hematopoiesis. Blood filtration is anatomically associated with the red pulp whilst detection of infectious particles and initiation of an immune response occurs via formation of germinal centers in the white pulp. The microanatomy of the human spleen, however, has been primarily extrapolated from extensive studies in rodents. Recent studies by Dr. Birte Steiniger (University of Marburg, Germany), have in fact indicated that as one example, the well- delineated B-cell compartment, known as the marginal zone, between white and red pulp is distinctly absent in the human spleen. The generation of a three dimensional (3D) tissue map of normal human spleen is therefore timely from both the scientific and pathologic perspectives. One of our obvious strengths is extensive experience with procurement and handling of transplant quality human organs using high level quality control measures (AIM I). Our proposed AIMs are designed to first obtain an understanding of the overall uniformity and macro-anatomy of the human spleen using MRI (AIM IIA). We have then designed interactive tissue handling (formalin-fixed paraffin embedded [FFPE], optimal cutting temperature [OCT] compound embedded, tissue clearing, and expansion) and optical microscopy (stochastic optical reconstruction microscopy [STORM], confocal, multiphoton, light sheet fluorescence microscopy [LSFM]) pipelines, which will resolve the microanatomy of the spleen from nanometer to millimeter in resolution (AIM IIB-D), superimposed with known spleen biomarkers. Isolated/dispersed splenocytes will be compared to peripheral blood mononuclear cells (PBMCs) from the same patient's blood (AIM III), and isolated immune cell subsets will be subjected to RNA- Seq (AIM IVB). Imaging mass cytometry (IMC) will provide the ultimate co-registration of biomolecules/biomarkers to individual cells (AIM IVA). Similarly, we will use multiplexed small molecule fluorescence in situ hybridization (FISH) and ultimately high throughput multiplexed error-robust FISH (MERFISH) to co-register mRNA expression to the cognate cell types (AIM IVB). Data from RNA-Seq of individual cells will further provide new splenic biomarkers to feed back to AIMs IIB-D and IVA. The ability to reconstruct an overall 3D spleen tissue map from all proposed pipelines is based on the common file format used for all optical microscopy, IMC and FISH applications as delineated in the Data Core. With all of these individual and cooperative strengths, we are poised to complete a 3D tissue map of the normal human spleen that can be shared with all HuBMAP, HIVE and TMC investigators and ultimately, the entire scientific community.